(R)-hydroxycarboxylic acid producing recombinant microorganism and process for preparing (r)-hydroxycarboxylic acid using the same

ABSTRACT

The present invention relates to recombinant microorganisms carrying both a gene encoding intracellular polyhydroxyalkanoate (PHA) depolymerase and a gene encoding PHA biosynthesis-related enzymes, and a process for preparing optically pure (R)-hydroxycarboxylic acids by culturing the recombinant microorganisms. The present invention provides: a recombinant microorganism transformed with two recombinant plasmids each of which contains a gene encoding intracellular PHA depolymerase and a gene encoding PHA biosynthesis-related enzymes, respectively; a recombinant microorganism harboring a gene encoding PHA biosynthesis-related enzymes in an integrated form with its chromosome, which is transformed with a recombinant plasmid containing a gene encoding intracellular PHA depolymerase; and, a process for preparing optically pure (R)-hydroxycarboxylic acids by culturing the recombinant microorganisms. In accordance with the present invention, (R)-hydroxycarboxylic acids can be released from the recombinant microorganisms into culture media after depolymerizing most PHA produced from the said microorganisms, which makes possible practical preparation of (R)-hydroxycarboxylic acids in a simple manner with reduced waste of substrates to increase the productivity, finally to allow the mass production of various optically pure (R)-hydroxycarboxylic acids.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to recombinant microorganisms producing(R)-hydroxycarboxylic acids and a process for preparing(R)-hydroxycarboxylic acids by employing the same, more specifically, torecombinant microorganisms carrying both a gene encoding intracellularPHA depolymerase and a gene encoding polyhydroxyalkanoate (PHA)biosynthesis-related enzymes, and a process for preparing optically pure(R)-hydroxycarboxylic acids by culturing the said recombinantmicroorganisms where biosynthesis and degradation of PHA occursimultaneously.

2. Description of the Prior Art

Since (R)-hydroxycarboxylic acid carries two functional groups, i.e.,hydroxyl group (—OH) and carboxyl group (—COOH), it is readily employedfor the organic syntheses of a variety of useful materials whileproviding chiral centers to the materials. Furthermore, the twofunctional groups can be converted readily into other forms, which makespossible its wide use of a chiral precursor compound in fine chemicalfields. In addition, (R)-hydroxycarboxylic acid may be used as apromising precursor for the syntheses of antibiotics, vitamins,aromatics and pheromones, nonpeptide ligand for the design of medicaland pharmaceutical products, and a precursor for novel pharmaceuticals,especially of carbapenem antibiotics which draw attentions as analternative of penicillin (see: Lee et al., Biotechnol. Bioeng.,65:363-368, 1999). For example, it has been reported that the method forsynthesis of (+)-thiennamycin from methyl-(R)-3-hydroxybutyrate wasdeveloped (see: Chiba and Nakai, Chem. Lett., 651-654, 1985).

On the other hand, polyhydroxyalkanoate (PHA), a polyester formed byester linkage of hydroxycarboxylic acids, is an energy and carbon sourcewhich is synthesized and accumulated in many species of microorganisms.Due to the optical specificity of its biosynthetic enzyme,hydroxycarboxylic acid as a monomer of PHA has only (R)-type opticalactivity, except for the compounds such as 4-hydroxybutyric acid whichhas no optical isomers. Accordingly, optically pure(R)-3-hydroxycarboxylic acids can be produced simply by depolymerizingthe biosynthesized PHA. On the ather hand, chemical processes forpreparing (R)-3-hydroxybutyrate, alkyl-(R)-3-hydroxybutyrate, oralkyl-(R)-3-hydroxyvalerate via depolymerization ofpoly-(hydroxybutyrate) (PHB) andpoly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) have been reportedin the art (see: Seebach et al., Org. Synth., 71:39-47, 1992; Seebachand Zuger, Helvetica Chim. Acta, 65:495-503, 1982). However, the saidprocesses for preparing (R)-3-hydroxybutyrate via chemical means haverevealed several disadvantages of low yield due to complicated steps ofculture of microorganism, recovery of cells, isolation of polymers,depolymerization and isolation/purification, and consumption of a largequantity of organic solvents. Moreover, a great deal of microbial cellmass are produced as a byproduct, which naturally limits trial ofproducing (R)-hydrocarboxylic acids merely to (R)-3-hydroxybutyrate and(R)-3-hydroxyvalerate.

Recently, the present inventors have reported a process for preparingvarious (R)-3-hydroxycarboxylic acids including (R)-3-hydroxybutyratevia autodegradation (depolymerization) employing PHA depolymerase, whichoccurs naturally with PHA biosynthesis-related enzymes in thePHA-producing microorganisms (see: Lee et al., Biotechnol. Bioeng.,65:363-368, 1999). The autodegradation method is more efficient thanconventional chemical methods, for example, after overproducing PHB inAlcaligenes latus by fermentation, incubation for 30 min under a properpH condition would allow the microorganism to degrade PHB into(R)-hydroxybutyrate with over 95% optical purity, which is then releasedinto a medium. Although the autodegradation method can be applied toproduce various (R)-3-hydroxycarboxylic acids, it essentiallyaccompanies a batch process in which PHA is accumulated and thendegraded. If biosynthesis and degradation of PHA could occursimultaneously in a continuous manner, it could be expected thatby-products of microbial materials can be dramatically reduced finallyto increase the yield of hydoxycarboxylic acids from the substrate. Inthis regard, there has been a continuing need to develop a process forpreparing (R)-3-hydroxycarboxylic acids by a simple continuous processto produce (R)-3-hydroxycarboxylic acids in a more efficient andeconomical way.

The general mechanism of biosynthesis and depolymerization of PHA in amicroorganism is as follows: When a microorganism is subject to anunbalanced growth condition of sufficient carbon source and limitedessential elements such as nitrogen, phosphate, or magnesium, it turnson the expression of the enzymes for PHA biosynthesis and thensynthesizes and accumulates PHA inside the cell body from excessivecarbon source through PHA biosynthesis (see: Lee, Biotechnol. Bioeng.,49:1-14, 1996). Later, when supply of essential elements are resumed,PHA is degraded into its monomer, (R)-3-hydroxycarboxylic acid, by theaction of PHA depolymerase and oligomeric hydrolysis enzymes (see:Muller and Seebach, Angew. Chem. Int. Ed. Engl., 32:477-502, 1993).

The mechanism for recycling of (R)-3-hydroxycarboxylic acid in themetabolic pathway whithin a microorganism has been established only for(R)-3-hydroxybutyrate as follows: By the action of (R)-3-hydroxybutyratedehydrogenase, (R)-3-hydroxybutyrate is converted into acetoacetatewhich is then recycled in the metabolic pathway of a microorganism (see:Muller and Seebach, Angew. Chem. Int. Ed. Engl., 32:477-502, 1993; Leeet al., Biotechnol. Bioeng., 65:363-368, 1999). Accordingly, both PHAbiosynthesis-related enzymes and PHA depolymerase are required for theproduction of (R)-3-hydroxycarboxylic acids in the microorganism, andfor the production of (R)-3-hydroxybutyrate, it is preferable to inhibitor remove (R)-3-hydroxybutyrate dehydrogenase activity.

The present inventors and many other researchers have conducted studieson the development of an efficient process for PHA production employingrecombinant E. coli, and in case of PHB or PHB/V, so favorable technicalprogress has been made to a level that products can be accumulated up toover 80% of total dried cell mass (see: Slater et al., J. Bacteriol.,170:4431-4436, 1988; Schubert et al., 170, 5837-5847, 1988; Kim et al.,Biotechnol. Lett., 14:811-816, 1992; Fidler and Dennis, FEMS Microbiol.Rev., 103:231-236, 1992; Lee et al., J. Biotechnol., 32:203-211, 1994;Lee et al., Ann. NY Acad. Sci., 721:43-53, 1994; Lee et al., Biotechnol.Bioeng., 44:1337-1347, 1994; Lee and Chang, J. Environ. Polymer Degrad.,2:169-176, 1994; Lee and Chang, Can. J. Microbiol., 41:207-215, 1995;Yim et al., Biotechnol. Bioeng., 49:495-503, 1996; Lee and Lee, J.Environ. Polymer Degrad., 4:131-134, 1996; Wang and Lee, Appl. Environ.Microbiol., 63:4765-4769, 1997; Wang and Lee, Biotechnol. Bioeng.,58:325-328, 1998; Lee, Bioprocess Eng., 18:397-399, 1998; Choi et al.,Appl. Environ. Microbiol, 64:4897-4903, 1998; Wong and Lee, Appl.Microbial. Biotechnol., 50:30-33, 1998; and, Lee et al., Int. J. Biol.Macromol., 25:31-36, 1999).

In nature, E. coli neither synthesize PHA as an intracellular storagematerial for energy nor have PHA depolymerase, and it is not consideredto have (R)-3-hydroxybutyrate dehydrogenase which converts(R)-3-hydroxybutyrate into acetoacetate. PHA-synthesizing recombinant E.coli, which is constructed by introducing genes encoding PHAsynthesis-related enzymes from other species, does not degrade theresulting PHA produced and accumulated in its cellular space because therecombinant E. coli carries only PHA biosynthesis-related enzymes (see:Lee, Trends Biotechnol., 14:98-105, 1996; Lee, Nature Biotechnol.,15:17-18, 1997). Therefore, the present inventors have perceived that(R)-3-hydroxycarboxylic acid, especially (R)-3-hydroxybutyrate can beproduced more efficiently by co-introducing/co-expressing the genes forPHA-synthesizing enzymes and PHA depolymerase in a recombinant E. coli,and furthermore, (R)-3-hydroxybutyrate would not be metabolized toacetoacetate in the absence of (R)-3-hydroxybutyrate dehydrogenase. Thisperception had inspired the present inventors to develop a recombinantmicroorganism co-transformed with genes for both PHAbiosynthesis-related enzymes and intracellular PHA depolymerase, and todevelop a process for preparing (R)-hydroxycarboxylic acids by employingthe same (see: Korean Patent Appln. No. 10-2000-0026158). However, theprocess for preparing (R)-hydroxycarboxylic acids provided by thepresent inventors has several disadvantages that it may increase theunit cost of production due to low productivity and a waste ofsubstrates resulting from the presence of undegradated PHA within themicroorganism after the culture.

Under the circumstances, there are strong reasons for developing aprocess for preparing optically active (R)-3-hydroxycarboxylic acids bydepolymerizing produced PHA into (R)-3-hydroxycarboxylic acids in a moreefficient manner while reducing the waste of substrates, finally toincrease the productivity of (R)-3-hydroxycarboxylic acids.

SUMMARY OF THE INVENTION

The present inventors have made an effort to develop an efficientprocess for preparing optically active (R)-3-hydroxycarboxylic acidswith increased productivity, and found that various optically pure(R)-hydroxycarboxylic acids can be prepared in a massive manner, byculturing microorganisms carrying both a gene encoding intracellular PHAdepolymerase and a gene encoding PHA biosynthesis-related enzymes, wherebiosynthesis and degradation of PHA occur simultaneously and thedepolymerization of almost all of the prepared PHA into(R)-hydroxycarboxylic acids is accomplished.

A primary object of the present invention is, therefore, to providerecombinant microorganisms producing optically pure(R)-hydroxycarboxylic acids.

The other object of the invention is to provide a process for preparingoptically pure (R)-3-hydroxycarboxylic acids by culturing the saidrecombinant microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present inventionwill become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

FIG. 1 is a genetic map of a recombinant plasmid of the invention,pUC19Red.

FIG. 2 is a genetic map of a recombinant plasmid of the invention,pUC19Red_stb.

FIG. 3 is a genetic map of a recombinant plasmid of the invention,p5184.

FIG. 4 is a graph showing the concentrations of dried-cell,polyhydroxybutyrate (PHB) and (R)-hydroxybutyrate monomer before andafter pyrolysis under a basic condition, the rates of PHB degradationand PHB biosynthesis, and the ratio of PHB degradation rate/PHBbiosynthesis rate, depending on the elapsed time during the culture of arecombinant E. coli of the invention, XL1-Blue/pSYL105Red.

FIG. 5 is a graph showing the concentrations of dried-cell, PHB and(R)-hydroxybutyrate monomer before and after pyrolysis under a basiccondition, the rates of PHB degradation and PHB biosynthesis, and theratio of PHB degradation rate/PHB biosynthesis rate, depending on theelapsed time during the flask culture of a recombinant E. coli of theinvention, XL1-Blue/pUC19Red;p5184.

FIG. 6 is a graph showing the concentrations of dried-cell, PHB and(R)-hydroxybutyrate monomer concentration before and after pyrolysisunder a basic condition, and pH, depending on the elapsed time duringthe batch culture of a recombinant E. coli of the invention,B-PHA+/pUC19Red_stb.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides: a recombinant microorganism transformedwith two recombinant plasmids each of which contains a gene encodingintracellular PHA depolymerase (SEQ ID No: 1) and a gene encoding PHAbiosynthesis-related enzymes (SEQ ID No: 3), respectively; a recombinantmicroorganism containing a gene encoding PHA biosynthesis-relatedenzymes in an integrated form with its chromosome, which is transformedwith a recombinant plasmid containing a gene encoding intracellular PHAdepolymerase; and, a process for preparing optically pure(R)-hydroxycarboxylic acids by culturing the recombinant microorganisms.The recombinant microorganism which is co-transformed with both a geneencoding intracellular PHA depolymerase and a gene encoding PHAbiosynthesis-related enzymes, carries a higher copy number of the geneencoding intracellular PHA depolymerase than that of PHAbiosynthesis-related enzymes. Although it is preferable that the saidgenes originate from Ralstonia eutropha, those of other species may beemployed in the invention. The gene encoding PHA depolymerase is presentin a plasmid in an inserted form, which may further containparB(hok/sok) locus originating from R1, E. coli plasmid. The geneencoding PHA biosynthesis-related enzymes may be present within a locusfor phosphotransacetylase (Pta) in an integrated form with itschromosome. A microorganism for transformation includes E. coli,preferably, E. coli XL1-Blue or E. coli B. Culture of the recombinantmicroorganism can be carried out by a continuous or batch process, where(R)-hydroxycarboxylic acids produced therefrom include(R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate in a monomeric or adimeric form.

The present invention is further illustrated in more detail as follows.

Using two plasmid vectors which can coexist in a microorganism with adifferent copy number, the present inventors introduced a gene encodingPHA biosynthesis-related enzymes into a plasmid of low copy number, andintroduced a gene encoding intracellular PHA depolymerase into a plasmidof high copy number, with an intention that the recombinantmicroorganisms transformed with the plasmids express a larger amount ofintracellular PHA depolymerase than PHA biosynthesis-related enzymes, toproduce and release hydroxycarboxylic acids including(R)-3-hydroxybutyrate without intracellular accumulation of PHA.Furthermore, based on the unexpected finding that the productivity andyield of (R)-hydroxycarboxylic acid was rapidly increased rather thandecreased when the expression level of PHA biosynthesis-related enzymeswas decreased, they tried to produce hydroxycarboxylic acids including(R)-3-hydroxybutyrate efficiently and release into liquid media, byculturing a recombinant E. coli transformed with a plasmid of high copynumber, which contains a gene encoding intracellular PHA depolymerase,while containing a gene encoding PHA biosynthesis-related enzymes in anintegrated form with its chromosome.

After all, the process of the present invention comprises a step ofpreparing optically pure (R)-hydroxycarboxylic acid released into theliquid media, by culturing a recombinant microorganism transformed withboth a recombinant plasmid of high copy number expressing intracellularPHA depolymerase and a recombinant plasmid of relatively low copy numberexpressing PHA biosynthesis-related enzymes, or, a recombinantmicroorganism transformed with a plasmid of high copy number expressingintracellular PHA depolymerase while containing a gene encoding PHAbiosynthesis-related enzymes in an integrated form with its chromosome.

In accordance with the present invention, the recombinant microorganismtransformed with both a gene encoding intracellular PHA depolymerase anda gene encoding PHA biosynthesis-related enzymes, is cultured usingglucose as a substrate, and produces monomers and dimers of(R)-3-hydroxybutyrate, which are then released into liquid media uponthe expression of the said enzymes. (R)-3-hydroxybutyrate and its dimersthus released can be separated on LC or HPLC under a specified analysiscondition. If required, the dimer can be degraded into(R)-3-hydroxybutyrate by way of heating it under a basic condition (see:Lee et al., Biotechnol. Bioeng., 65:363-368, 1999).

The present inventors isolated a DNA fragment (SEQ. ID No: 1) includinga gene encoding intracellular PHA depolymerase and its ribosome-bindingsite by means of polymerase chain reaction (PCR) with reference to DNAsequence from Ralstonia eutropha accessible on GenBank (see: Saito andSaegusa, GenBank Sequence Database, AB017612, 2001; Saegusa et al., J.Bacteriol., 183:94-100, 2001), and then introduced the said fragmentinto pUC19 (New England Biolabs, USA) of high copy number, finally toprepare pUC19Red. Further, for the purpose of increasing the stabilityof a plasmid, pUC19Red was introduced by a DNA fragment containingparB(hok/sok) locus (SEQ. ID No. 2) (see: GenBank Sequence Data, X05813;Gedes, Bio/Technology, 6:1402-1405, 1988) to give pUC19Red-stb, wherethe DNA fragment was obtained by the digestion of pSYL105(see: Lee etal., Biotechnol. Bioeng., 44: 1337-1347, 1994) with the restrictionendonuclease EcoRI and BamHI.

Meanwhile, a DNA fragment (SEQ. ID No: 3) containing a gene for operonwhich comprises PHA biosynthesis-related enzymes from Ralstoniaeutropha, including PHA synthase (PhaB), β-ketothiolase (PhaA) andreductase (PhaB), and a DNA fragment containing parB(hok/sok) locus wereobtained by digestion of pSYL105 (see: Lee et al., Biotechnol. Bioeng.,44: 1337-1347, 1994) with restriction endonuclease XbaI, which were thenintroduced into XbaI site of pACYC184 (New England Biolabs, USA) capableof coexisting with plasmids derived from pUC19, finally to constructp5184.

Further, to prepare a microorganism harboring a gene encoding PHAbiosynthesis-related enzymes in an integrated form with its ownchromosome, a gene encoding PHA biosynthesis-related enzymes fromRalstonia eutropha, which was obtained by digestion of pSYL105 (see: Leeet al., Biotechnol. Bioeng., 44:1337-1347, 1994) with restrictionendonuclease BamHI, was introduced within phosphotransacetylase (Pta)locus in the chromosome of E. coli B (ATCC 11303) by means of homologousrecombination, and prepared E. coli B-PHA+, a mutant E. coli for PHAbiosynthesis.

E. coli XL1-Blue (Stratagene Cloning System, USA) was transformed withthe said pUC19Red and p5184 through electroporation, to prepare arecombinant E. coli XL1-Blue/pUC19Red;p5184, which contains a geneencoding PHA biosynthesis-related enzymes and a gene encodingintracellular PHA depolymerase. Then, by transforming the said E. coliB-PHA+ with said pUC19Red_stb through electroporation, a recombinant E.coli B-PHA+/pUC19Red_stb having both a gene encoding PHAbiosynthesis-related enzymes and a gene encoding intracellular PHAdepolymerase was prepared. Each of the recombinant microorganisms wascultured in media containing proper carbon source, then theconcentration of (R)-hydroxybutyrate produced therefrom was measured,respectively. As a result, it was found that (R)-hydroxybutyrate can beproduced efficiently by culturing each of the said recombinantmicroorganisms, and appropriate conditions for their culture wasestablished. For the production of (R)-3-hydroxybutyrate, a recombinantmicroorganism co-transformed with both a gene encoding intracellular PHAdepolymerase from Ralstonia eutropha and a gene encoding PHAbiosynthesis-related enzymes from Alcaligenes latus or Ralstoniaeutropha, is preferably cultured for 30 to 70 hours.

Although the present invention employed two plasmid vectors of pUC19with pACYC184 that can coexist with each other, it is obvious for thoseskilled in the art that any combination of the vectors that can coexistwould be applicable in the invention. For the genes encoding PHAbiosynthesis-related enzymes and intracellular PHA depolymerase, it isobvious for those skilled in the art that genes for the same role fromany microorganism other than Ralstonia eutropha is similarly applicableif the gene can be active in host microorganism. In addition, it isobvious for those skilled in the art that for chromosomal integration ofa gene encoding PHA biosynthesis-related enzymes, it can be incorporatedwithin any locus other than phosphotransacetylase locus, as far as itdoes not have a critical influence on the metabolism of themicroorganism. Moreover, it is obvious for those skilled in the art thathost microorganisms for the expression of the plasmids can be any E.coli strain other than E. coli XL1-Blue, and that for chromosomalintegration of a gene encoding PHA biosynthesis-related enzymes, any E.coli strain other than E. coli B may be applicable if it is able to havean altered chromosome. Further, it is obvious for those skilled in theart that a host microorganism of the present invention can be anymicroorganism being capable of expressing the said enzymes other than E.coli.

The present invention is further illustrated in the following examples,which should not be taken to limit the scope of the invention.

EXAMPLE 1 Cloning of a Gene Encoding Intracellular PHA Depolymerase fromRalstonia eutropha

For the cloning of intracellular PHA depolymerase from Ralstoniaeutropha, PCR was performed by using a chromosomal DNA isolated fromRalstonia eutropha according to Marmur et al's method (see: Marmur, J.Mol. Biol., 3:208-218, 1961) as a template and using primer1,5′-GCTCTAGAGGATCCTTGTTTTCCGCAGCAACAGCT-3′ (SEQ. ID No: 4)) and primer2,5′-CGGGATCCAAGCTTACCTGGTGGCCGAGGC-3′ (SEQ. ID No: 5), complementary tothe sequence of a gene encoding intracellular PHA depolymerase fromRalstonia eutropha (see: Saito and Saegusa, GenBank Sequence Database,AB017612, 2001). In carrying out the PCR, pre-denaturation was firstmade at 95° C. for 5 min and then, 30 cycles of reactions wereperformed, each of which comprises the steps of: denaturation at 95° C.for 50 sec; annealing at 55° C. for 1 min 10 sec; and, elongation at 72°C. for 3 min. Finally, post-elongation was made at 72° C. for 7 min. Theresulting DNA fragment was digested with a restriction endonucleaseBamHI, and 1.4 kbp of DNA fragment was fractionated on agarose gelelectrophoresis, and ligated into pUC19 (see: Sambrook et al., MolecularCloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; New England Biolabs, USA)digested with the same restriction endonuclease, to construct pUCRed.Then, E. coli XL1-Blue was transformed with pUC19Red by the technique ofelectroporation and then, was screened for successful transformants onLB plate media (Yeast extract, 5 g/L; tryptone, 10 g/L; NaCl, 10 g/L)containing ampicillin (50 mg/L) and bacto-agar (15 g/L), finally toprepare a recombinant microorganism of XL1-Blue/pUC19Red. Comparisonwith the sequences accessible on GenBank™ revealed that sequence of thecloned DNA fragment is a gene encoding intracellular PHA depolymerasefrom Ralstonia eutropha including its innate promoter region forconstitutive expression and its ribosome-binding site. FIG. 1 is agenetic map of pUC19Red, a recombinant plasmid of the present invention.

Then, the digestion of pSYL105 with restriction endonuclease EcoRI andBamHI was performed to obtain a DNA fragment including parB(hok/sok)locus (see: Gedes, Bio/Technology, 6:1402-1405, 1988), which was thenintroduced into pUC19Red to construct pUC19Red_stb. FIG. 2 is a geneticmap of pUC19Red_stb, a recombinant plsmid of the present invention.

EXAMPLE 2 Construction of a Plasmid Containing a Gene Encoding PHABiosynthesis-Related Enzymes from Ralstonia eutropha, which can Coexistwith Other Plasmids in E. coli

Since pUC19Red and pUC19Red_stb prepared in Example 1 can express onlythose enzymes related to depolymerization of intracellular PHAs intomonomers, E. coli should be transformed with another plasmid containinga gene encoding PHA biosynthesis-related enzymes to prepare arecombinant microorganism producing (R)-3-hydroxybutyrate. To this end,a plasmid p5184 containing a gene encoding PHA biosynthesis-relatedenzymes from Ralstonia eutropha and being capable of coexisting withother plasmids derived from pUC19 containing ColE1-compatible origin ofreplication, was prepared as follows: First, pSYL105 was digested withrestriction endonuclease XbaI, then subject to agarose gelelectrophoresis to obtain 5.6 kbp of DNA fragment containing a geneencoding PHA biosynthesis-related enzymes from Ralstonia eutropha andhok/sok gene. Then, the DNA fragment was incorporated into pACYC184 (NewEngland Biolabs, USA) having a p15A origin of replication, which wasdigested with the same restriction endonuclease, finally to preparep5184. FIG. 3 is a genetic map of p5184, a recombinant plasmid of thepresent invention.

EXAMPLE 3 Preparation of E. coli Harboring a Gene Encoding PHABiosynthesis-Related Enzymes in an Integrated Form with its Chromosome

To insert a gene encoding PHA biosynthesis-related enzymes within E.coli phosphotransacetylase (Pta) locus by way of homologousrecombination (see: Yu et al., Proc. Natl. Acad. Sci., USA,97:5978-5983, 2000), PCR was carried out for the amplification of a geneencoding phosphotransacetylase with two halves of fragments, by using achromosomal DNA of E. coli B (ATCC 11303) as a template and using primer3,5′-GCGAATTCTTTAAAGACGCGCGCATTTCTAAACT-3′ (SEQ ID No: 6), primer 4,5′-GCGGTACCGAGCTCCGGGTTGATCGCACAGTCA-3′ (SEQ ID No: 7), primer5,5′-GGCGAGCTCGCGCATGCCCGACCGCTGAACAGCTG-3′ (SEQ ID No: 8) and primer6,5′-GCAAGCTTTTTAAAGCGCAGTTAAGCAAGATAATC-3′ (SEQ ID No: 9). And, one ofthe half fragment was digested with restriction endonuclease EcoRI andKpnI while the other half fragment was digested with SphI and HindIII,and then were ligated into a pUC19 plasmid digested with the samerestriction endonucleases, respectively. A kanamycin resistance gene wasamplified by PCR using pACYC177(New England Biolabs, USA) as a templateand using primer 7,5′-GCTCTAGAGAGCTCAAAGCCACGTTGTGTCTCAAA-3′ (SEQ ID No:10) and primer 8,5′-GCGCATGCTTAGAAAAACTCATCGAGCATC-3′ (SEQ ID No: 11),and the resulting fragment was digested with SalI and SphI and thenligated into a plasmid containing the said two fragments of pta gene. Agene fragment including a gene encoding PHA biosynthesis-relatedenzymes, which was prepared from the digestion of pSYL105 withrestriction endonuclease BamHI, was also ligated into BamHI site of theplasmid. Then, the plasmid was digested with restriction endonucleaseDraI and then the resulting fragments including all the said genes werefractionated on agarose gel electrophoresis.

E. coli B (ATCC 11303) transformed with pTrcEBG (see: Korean PatentAppln. No. 10-2001-48881), was induced by IPTG to be used for competentcell, which was then transformed with the said fragments byelectroporation, and then, the colonies whose chromosomes wereintegrated with the said fragments were selected on Luria-Bertani (LB)plate media containing kanamycin. Then, colonies lacking pTrcEBG wereselected as a result of subculture in LB liquid media containingkanamycin, and PCR amplification of the gene encoding PHAbiosynthesis-related enzymes using chromosomal DNA as a template, wasfollowed to confirm the correct integration of the gene into chromosome.PHA synthesis through the culturing them in media containing glucose,made sure that PHA biosynthesis-related enzymes have a biologicalactivity. Then, the selected recombinant E. coli was named as ‘E. coliB-PHA+’.

EXAMPLE 4 Preparation of a Recombinant Microoranism Producing(R)-hydroxycarboxylic Acids

E. coli XL1-Blue was transformed with both pUC19Red and p5184 preparedin Examples 1 and 2 by way of electroporation to prepare recombinant E.coli XL1-Blue/pUC19Red;p5184. And, E. coli B-PHA+ prepared in Example 3was transformed with pUC19Red_stb prepared in Example 1 byelectroporation to prepare recombinant E. coli B-PHA+/pUC19Red_stb.

EXAMPLE 5 Preparation of (R)-hydroxybutyrate

Recombinant E. coli XL1-Blue/pSYL105Red (see: Korean Patent Appln. No.10-2000-002615) was cultured in LB media containing 50 mg/L ampicillinfor 12 hours, and then 1 in Q of the culture was inoculated in 250 mlflask containing 100 ml of R media with 20 g/L of glucose, 20 mg/L ofthiamin and 50 mg/L of ampicillin (see: Choi et al., Appl. Environ.Microbiol., 64:4897-4903, 1998), and then cultured at 37° C. in ashaking incubator at 250 rpm. Then, the recombinant E. coliXL1-Blue/pUC19Red;p5184 was cultured for 12 hours in LB media containing50 mg/L of ampicillin and 50 mg/L of chlorampenicol and then 1 ml of theculture was inoculated in 250 ml flask containing 100 ml of R media with20 g/L of glucose, 20 mg/L of thiamin, 50 mg/L of ampicillin and 50 mg/Lof chloramphenicol (see: Choi et al., Appl. Environ. Microbiol.,64:4897-4903, 1998), and was cultured at 37° C. in a shaking incubatorat 250 rpm. FIG. 4 is a graph showing the concentrations ofdried-microorganism (●), polyhydroxybutyrate (PHB) (▾) and PHB monomersprepared before (∇) and after () pyrolysis under a basic condition, therates of PHA biosynthesis (

) and PHB degradation (⋄), and the ratio of PHB degradationrate/biosynthesis rate (▴), depending on the elapsed time during theculture of recombinant E. coli XL1-Blue/pSYL105Red. FIG. 5 is a graphshowing the concentrations of dried-microorganism (●), PHB (▾) and PHBmonomers prepared before (∇) and after () pyrolysis under a basiccondition, and the rates of PHA biosynthesis (

) and PHB degradation (⋄), and the ratio of PHB degradationrate/biosynthesis rate (▴), depending on the elapsed time during theflask culture of recombinant E. coli XL1-Blue/pUC19Red;p5184. As can beseen in the results of FIGS. 4 and 5, it was demonstrated that:recombinant E. coli XL1-Blue/pUC19Red;p5184 showed rather rapid PHAbiosynthesis than expectation that expression level of the PHAbiosynthesis-related enzymes is lowered due to relatively low copynumber of its plasmid; and, the rate of PHA degradation became high. IfPHA is degradated into a form smaller than dimeric form, it may bereleased out of a microorganism without difficulty. Therefore,(R)-hydroxybutyrate was prepared mostly in a dimeric form, which was, inturn, easily changed into a monomeric form by pyrolysis under a basiccondition (see: Lee et al., Biotechnol. Bioeng., 65:363-368, 1999;Korean Patent No. 250830; PCT International Appln. No. PCT/KR98/00395).

In addition, the recombinant E. coli B-PHA+/pUC19Red_stb prepared inExample 4 was cultured in a batch-wise in 2.5 L jar fermentor(KoBiotech, Korea) containing 1.5 L of R media (initial pH 7.0) with 20g/L of glucose (see: Choi et al., Appl. Environ. Microbiol.,64:4897-4903, 1998), at 37° C. in a shaking incubator at 500 rpm whilenot adjusting pH during the culture. FIG. 6 is a graph showing theconcentrations of dried-microorganism (●), PHB (▾) and PHB monomersprepared before (∇) or after () pyrolysis under a basic condition andpH (▮) depending on the elapsed time during the batch culture of arecombinant E. coli B-PHA+/pUC19Red_stb of the present invention. As canbe seen in FIG. 6, 34 hours of culture allowed the total production of11.8 g/L (R)-3-hydroxybutyrate and its dimers, which is estimated to bea final yield of up to 59%. Further, it was examined that the plasmidswere stable enough since no microorganism lacking its plasmid was foundduring the culture, indicating that there is no need to use antibioticsfor maintaining its plasmid.

EXAMPLE 6 Simultaneous Production of (R)-hydroxybutyrate and(R)-hydroxyvalerate

The recombinant E. coli XL1-Blue/pUC19Red_stb was cultured for 12 hoursin LB media, and 1 ml of the culture was inoculated in 250 ml flaskcontaining 100 ml of R media with 10 g/L of glucose and 1 g/L ofpropionic acid 20 mg/L (see: Choi et al., Appl. Environ. Microbiol.,64:4897-4903, 1998), and then cultured at 37° C. in a shaking incubatorat 250 rpm. After 48 hours of culture, pyrolysis was performed under abasic condition, and HPLC analysis of the resultant revealed that 4.1g/L of (R)-3-hydroxybutyrate and 0.4 g/L of (R)-3-hydroxybutyrate wereprepared, respectively.

As clearly illustrated and demonstrated as above, the present inventionprovides recombinant microorganisms carrying both a gene encodingintracellular PHA depolymerase and a gene encoding polyhydroxyalkanoate(PHA) biosynthesis-related enzymes, and a process for preparingoptically pure (R)-hydroxycarboxylic acids by culturing the saidrecombinant microorganisms where biosynthesis and degradation of PHAoccur simultaneously. In accordance with the present invention,(R)-hydroxycarboxylic acids can be released from the recombinantmicroorganisms into culture media after depolymerizing most PHA producedfrom the said microorganisms, which makes possible practical preparationof (R)-hydroxycarboxylic acids in a simple manner with reduced waste ofsubstrates to increase the productivity, finally to allow the massproduction of various optically pure (R)-hydroxycarboxylic acids.

While the present invention has been shown and described with referenceto the particular embodiments, it will be apparent to those skilled inthe art that many changes and modifications may be made withoutdeparting from the spirit and scope of the invention as defined in theclaims.

1. A recombinant microorganism producing optically pure(R)-hydroxycarboxylic acid, which is co-transformed with a recombinantplasmid containing a gene encoding intracellular polyhydroxyalkanoate(PHA) depolymerase (SEQ ID No: 1) and a recombinant plasmid containing agene encoding PHA biosynthesis-related enzymes.
 2. The recombinantmicroorganism of claim 1, wherein the gene encoding intracellular PHAdepolymerase and the gene encoding PHA biosynthesis-related enzymes arederived from Ralstonia eutropha.
 3. The recombinant microorganism ofclaim 1, wherein the copy number of the gene encoding PHA depolymeraseis higher than that of the gene encoding PHA biosynthesis-relatedenzymes.
 4. The recombinant microorganism of claim 1, wherein themicroorganism is Escherichia coli.
 5. The recombinant microorganism ofclaim 1, wherein the microorganism is E. coli XL 1-Blue.
 6. Therecombinant microorganism of claim 1, wherein (R)-hydroxycarboxylic acidis (R)-3-hydroxybutyrate or (R)-3-hydroxyvalerate.
 7. The recombinantmicroorganism of claim 6, wherein (R)-3-hydroxybutyrate and(R)-3-hydroxyvalerate is in monomeric or dimeric form.
 8. Escherichiacoli XLI-Blue/pUC19Red;p5184 producing optically pure(R)-hydroxycarboxylic acid.
 9. A recombinant microorganism producingoptically pure (R)-hydroxycarboxylic acid, which is transformed with arecombinant DNA encoding intracellular PHA depolymerase while containinga gene encoding PHA synthesis-related enzymes in an integrated form withits chromosome.
 10. The recombinant microorganism of claim 9, whereinthe gene encoding PHA synthesis-related enzymes is present withinphosphotransacetylase (Pta) locus in an integrated form with itschromosome.
 11. The recombinant microorganism of claim 9, wherein thegene encoding intracellular PHA deploymerase is present in a plasmid inan inserted form.
 12. The recombinant microorganism of claim 9, whereinthe gene encoding intracellular PHA depolymerase is present in a plasmidtogether with parB(hok/sok) locus (SEQ ID No: 10) derived from E. coliR1 in an inserted form.
 13. The recombinant microorganism of claim 9,wherein the gene encoding intracellular PHA depolymerase and the geneencoding PHA biosynthesis-related enzymes are derived from Ralstoniaeutropha.
 14. The recombinant microorganism of claim 9, wherein themicroorganism is Escherichia coli.
 15. The recombinant microorganism ofclaim 9, wherein the microorganism is E. coli XL1-Blue or E. coli B. 16.The recombinant microorganism of claim 9, wherein (R)-hydroxycarboxylicacid is (R)-3-hydroxybutyrate or (R)-3-hydroxyvalerate.
 17. Therecombinant microorganism of claim 16, wherein (R)-3-hydroxybutyrate and(R)-3-hydroxyvalerate are monomeric or dimeric form.
 18. A recombinantmicroorganism, E. coli B-PHA+/pUC19Red_stb producing optically pure(R)-hydroxycarboxylic acid.
 19. A process for preparing optically pure(R)-hydroxycarboxylic acid, which comprises the steps of culturing therecombinant microorganism of claim 1 and obtaining (R)-hydroxycarboxylicacid from the culture.
 20. The process of claim 19, wherein theculturing is carried out by continuous or batch process.
 21. The processof claim 19, wherein (R)-hydroxycarboxylic acid is (R)-3-hydroxybutyrateor (R)-3-hydroxyvalerate.
 22. The process of claim 21, wherein(R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate are monomeric or dimericform.
 23. A process for preparing optically pure (R)-hydroxycarboxylicacid, which comprises the steps of culturing the recombinantmicroorganism of claim 9 and obtaining (R)-hydroxycarboxylic acid fromthe culture.
 24. The process of claim 23, wherein the culturing iscarried out by continuous or batch process.
 25. The process of claim 23,wherein (R)-hydroxycarboxylic acid is (R)-3-hydroxybutyrate or(R)-3-hydroxyvalerate.
 26. The process of claim 25, wherein(R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate are monomeric or dimericform.
 27. A process for preparing optically pure (R)-hydroxycarboxylicacid, which comprises the steps of culturing E. coliXL1-Blue/pUC19Red;p5184 and obtaining (R)-hydroxycarboxylic acid fromthe culture.
 28. A process for preparing optically pure(R)-hydroxycarboxylic acid, which comprises the steps of culturing E.coli B-PHA+/pUC19Red_stb and obtaining (R)-hydroxycarboxylic acid fromthe culture.